Axonal regeneration after optic nerve crush in Nogo-A/B/C knockout mice

Purpose The axonal regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) crush was investigated both in vivo and in vitro on Nogo-A/B/C knockout mice. Methods The study used 20 Nogo-A/B/C knockout mice in the experimental group, and 20 C57BL/6 mice in the control group. Partial ON injury was induced by using a specially designed ON clip to pinch the ON 1 mm behind the mouse eyeball with 40 g pressure for 9 s. The left ON was injured in both groups, but the right ON was left untouched in the control group. Nogo-A/B/C mRNA was studied by in situ hybridization in both groups. GAP-43 was studied by immunofluorescence staining on frozen sections. RGCs were purified and cultured in DMEM medium containing B-27. Cells were then immunostained with both Thy1.1 and GAP-43 antibodies. The axonal growth of RGCs was calculated by a computerized image analyzer. Results GAP-43 expression was significantly higher in the experimental group than in the control group (p<0.01). GAP-43 antibody binding was demonstrated in the axons of cultured RGCs. Axonal growth was significantly more active at every observed time point in the experimental group than in the control group (F=43.25, 32.16; p<0.01). Conclusions Nogo genes play an inhibitive role in the axonal regeneration after ON injury, while Nogo-knockout is an effective way to eliminate this inhibition and accelerate axonal regeneration.

Generation and maintenance of Nogo-A/B/C knockout mutant mice: A Nogo-targeted embryonic stem cell clone was identified from the Omnibank sequence Tag database. Omnibank mutations were created, using insertional mutagenesis based on retroviral-based gene trap methodology. Heterozygotic mice harboring the disrupted allele were bred to C57Bl/6 mice for expansion in our animal housing facility and intercrossed to maintain the Nogo-A/B/C mutation on a hybrid 129SvEvBrd_C57Bl/6 background. The heterozygotic mice (9 or 12-week-old) were donated by Professor Xiulan Xu. The mice were raised in the laminar air flow cage rack (Suzhou Experimental Equipment Company, Suzhou, China). In the present study, animals were backcrossed to C57Bl/6 for three to six generations, and 7-14-week-old Nogo-A/B/C -/-/littermates were used as controls for all experiments. The present investigation adhered to the principles regarding the care and use of animals according to the American Physiologic Society, the Society for Neuroscience, and the ARVO State-ment for the use of animals in ophthalmic and vision research.
Animal grouping: The present study used 20 Nogo-A/B/ C knockout mice (8 or 12-week-old) in the experimental group, and 20 C 57BL/6 control mice (8 or 12-week-old) in the control group. To depict the axonal growth at successive time points, both groups were further divided into three subgroups representing the first day (n=7), third day (n=7), and seventh day (n=7) outcomes, respectively.
Surgical procedure: Prior to ON crush, general anesthesia was induced in each animal with an intraperitoneal injection of 1% sodium pentobarbital solution (50 mg/kg body weight). A surgical microscope was used to visualize the ON, which was exposed through a superior temporal approach. A 1-1.5 cm incision was made in the skin above the right orbit. The dural sheath of the ON was opened longitudinally, from which the ON was then gently detached before it was crushed at 1 mm distal to the eyeball by an ON forceps with 40 g pressure for 9 s. Injury to the ophthalmic artery was carefully 269 avoided during the procedure. Nerve injury was verified by the appearance of a clearing at the crush site; the vascular integrity of the retina was verified by funduscopic examination after dilating the pupil with atropine. The left ON was crushed in both groups, but the right ON was left untouched in the control group.
Preparation for pathological and immunohistochemistry examinations: Seven days after ON crush, thoracotomy was performed. The animals were perfused through the heart with heparin saline followed by 4% paraformaldehyde, including diethypyrocarbonate (DEPC) for in situ hybridization. Eyes with ON segments up to the optic chiasm were postfixed overnight, and transferred to a 30% sucrose solution overnight (4°C ) and including DEPC for in situ hybridization overnight (4°C ). Frozen sections (15 µm) were cut longitudinally on a cryostat, thaw mounted onto coated glass slides, and stored at -80 °C until further use.
Digoxygenin-labeled sense and antisense RNA probes were generated as described [5]. The serial number (NM-57142) and cDNA array of Nogo gene was achieved from GenBank. The probe was designed with Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA). The common probe, recognizing all three isoforms of Nogo, contains transcript A sequence between nucleotides 2535 and 4678.
Cryostat sections (15 µm) were mounted on Superfrost-Plus slides. Sections were post-fixed in 4% paraformaldehyde and PBS, acetylated in 0.1 M triethanolamine and 0.25% acetic anhydride, and permeabilized for 20 min in 1% Triton X-100 and PBS. Hybridization was performed overnight in 5x300 mM NaCl, 30 mM sodium citrate, pH 7.0 (SSC) buffer containing 50% formamide and 2% blocking reagent at 68 °C. Two rigorous washes were performed in 0.2X SSC at the same temperature for 1 h, and signals were detected with alkaline phosphatase-coupled anti-digoxigenin antibodies using spreptavidin-biotin-peroxidase complex (SABC) as color reaction substrates.
Isolation and culture of retinal ganglion cells: The eyes of the mice were collected after they were given a lethal dose of sodium pentobarbital. Retinas were isolated and dissected under an anatomic microscope to remove visible vessels before they were washed three times in Hank's balanced salt solution.
RGCs from the retina were isolated and purified as previously described [6,7]. Briefly, the retinal tissue was dissociated into single cells in Eagle's minimum essential medium that contained 15 U/ml papain and 70 U/ml collagenase. The cell suspension was incubated successively in a polypropylene tube coated with a monoclonal anti-rat macrophage IgG to remove macrophages, and then in another one coated with a monoclonal anti-rat Thy 1.1 IgG. Finally, the tube was gently washed five times with PBS. RGCs were collected by centrifugation at 700xg for 5 min and then cultured at a concentration of 6x10 6 cells/ml in DMEM that contained 100 U/ml penicillin, 100 µg/ml gentamicin, and B-27. Cells were then seeded into poly-lysine coated 24-well plates and incubated at 37 °C with 5% CO 2 ventilation. Half of the medium was renewed with cytarbine (20 µg/ml) added at certain intervals.    Cells were examined daily under phase-contrast microscope.
Immunocytochemistry: RGCs were fixed in 4% paraformaldehyde for 15 min and blocked with 5% goat serum. After three washes with PBS, the cells were incubated successively with primary antibody anti-GAP-43 (1:1000) at 4 °C for 24 h, secondary antibody goat-anti-rabbit IgG at 37 °C for 1 h, and SABC at 37 °C for 30 min. Between each step, cells were washed three times with PBS. After coloration with DAB, dehydration, and dimethyl benzene treatment, slides were mounted and examined with phase-contrast microscopy.
Statistical analysis: The data were analyzed by the twotailed Student t-test, using software (Origin ver. 6.0, OriginLab Corp., Northampton, MA). Expression of Nogo-A/B/C was calculated using image analysis software. The images were taken using a digital camera mounted on a microscope. The analysis was performed using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD). Positvie area of stain of Nogo-A/B/C expression of experimental group was 1.36±0.05x10 2 µm/m 2 , whereas that of control group was 26.56±1.25x10 2 µm/m 2 . There was significant difference positvie area of stain of Nogo-A/B/C expression between experimental group and control group (p<0.01). Little or no expression of Nogo-A/B/C mRNA was observed in the ON of Nogo-A/B/C-knockout mice ( Figure 1A). Expression of Nogo-A/B/C mRNA was demonstrated in the ON of young adult normal mice ( Figure 1B There was a significant difference in the size of the areas that stained positively for GAP-43 on the ON sections between experimental(Nogo-A/B/C knockout mice) and control groups (C57BL/6 control mice; p<0.01; Table 1). The positive area of GAP-43 stain of the experimental group increased significantly from one day to seven days after crush. There was little increase of the positive area of GAP-43 stain of the control group.

Expression of Nogo-
In vitro axonal growth of the experimental and control groups: Adherence of RGCs began at 12 h after they were seeded into poly-lysine-coated 24-well plates and incubated at 37 °C 5% CO 2 ventilation, and finished in 24 h. After this, a single layer of round and oval RGCs was seen on the plates.
We investigated axonal generation by staining of GAP-43. Axonal length was longer in the third day for the experimental group (Nogo-A/B/C knockout mice; Figure 3A) than that of the control group (C57BL/6 control mice) of the same day ( Figure 3B).  Table 2). The scale bars represent 40 µm (GAP-43 stain). The difference in axonal length between experimental group (Nogo-A/B/C knockout mice) increased from the first day to seventh day after crush. The axonal length of control group (C57BL/6 control mice) increased from the first day to third day, then descreased from the third day to seventh day. The difference in axonal length between experimental group (Nogo-A/B/C knockout mice) and control group (C57BL/6 control mice) was not significant (*p>0.05) on the first postoperative day. However, the difference in axonal length was significant by the third and seventh days between experimental group and control group.

TABLE 2. AXONAL LENGTH OF RETINAL GANGLION CELLS OF DIFFERENT
Axonal length was calculated using image analysis software. The images were taken using a digital camera mounted on a microscope. For neurite length determinations, the neurites were traced and the morphometry analysis was performed using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD). The length of the axon was defined as the distance from the soma to the tip of the process. An average of 50 neurons from each group was selected from a number of chamber slides. The difference in axonal length of the experimental group (Nogo-A/B/C knockout mice) increased from the first day to the seventh day after crush. The axonal length of the control group (C57BL/6 control mice) increased from the first day to the third day, then decreased from the third day to the seventh day. The difference in axonal length between the experimental group (Nogo-A/B/C knockout mice) and the control group (C57BL/6 control mice) was not significant (p>0.05) on the first postoperative day. However, the difference in axonal length became obvious by the third and seventh days between the experimental and control groups. (p<0.01; Table  2).
The Nogo gene has been cloned and its product appears to be a new member of the reticulon family [12]. An extracellular segment composed of 66 amino acid residues between two transmembrane domains, known as Nogo-66, is the major functional domain of this protein [3].
A neutralizing monoclonal antibody, neurite outgrowth inhibitor 1 (IN-1), has recently been confirmed to target the N-terminal domain of Nogo-A encoded by exon 3 [13]. When IN-1 is applied to several CNS injury models, including spinal cord lesions and cortical lesions, there is notable neuroanatomical and behavioral recovery compared with controls [14]. These results suggest that the Nogo-NgR system is accountable for, to a large degree, the inhibitory activity that is responsible for the prevention of CNS axonal regeneration after injury.
Increased expression of Nogo-A in RGCs after ON injury indicates that Nogo-A may play an inhibitory role in axonal regeneration after ON injury.
The expressed GAP-43 is located in the growth cone of the regenerating axon. Positive expression of GAP-43 in our figures indicate regeneration of axon of optic neve.
There was a significant difference in the area of ON stained positive for GAP-43 between the experimental group(Nogo-A/B/C knockout mice) and the control group(C57BL/6 mice) in this study. Nogo is important inhibitor for regeneration after CNS injury. Knockout of Nogo-A/B/C gene can lead to regeneration after optic nerve injury in this study. The results indicate that Nogo-A/B/C plays an important role in the inhibition of axonal regeneration after ON injury.
The significant difference in the length of axons regenerated in vitro between the experimental and control groups on the third and seventh postoperative days suggests that Nogo-A/B/C elimination is effective in enhancing axonal regeneration of RGCs in at least in vitro experiments.
The analysis on mice with various degrees of gene disruption targeted at Nogo isoforms has been reported by three groups. Kim et al. [11] observed significant corticospinal tract fiber sprouting in young Nogo-A/B -/mice compared with wildtype and Nogo-A/B +/mice. Simonen et al. [15] noted moderate corticospinal tract fiber regeneration in Nogo-A -/mice. However, Zheng et al. [16] saw no significant regeneration in either Nogo-A/B -/line or Nogo-A/B/C -/line mice above that of wild type. It is believed that the varying degrees of regenerative capacity after Nogo elimination can be attributed to the differences in genetic backgrounds of the mice used in the experiments [17].
Increased expression of Nogo-A in RGCs after ON injury indicates that Nogo-A may play an inhibitory role in axonal regeneration after ON injury.
There was a significant difference in the area of ON stained positive for GAP-43 between the experimental group and the control group in this study. The expressed GAP-43 is located in the growth cone of the regenerating axon. The results indicate that Nogo-A/B/C plays an important role in the inhibition of axonal regeneration after ON injury.
The significant difference in the length of axons regenerated in vitro between the experimental and control groups on the third and seventh postoperative days suggests that Nogo-A/B/C elimination is effective in enhancing axonal regeneration of RGCs in at least in vitro experiments.
Several myelin inhibitors of axonal growth, including Nogo-66, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, exert their effects through NgR. Further investigation of NgR may reveal whether NgR gene knockout enhances axonal regeneration after ON injury even more effectively.